This SHARMA_DATASET_readme.txt file was generated on 2021-03-06 by Govinda Sharma

GENERAL INFORMATION

Journal: Submitted to f1000research.

Title: Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss.

Author(s): 
Govinda Sharma1, Rasha Saubouny1, Matthew M Joel2,3, Kristina Martens2, Davide Martino4, A.P. Jason de Koning3, Gerald Pfeffer2*, Timothy E. Shutt1*
1 Departments of Medical Genetics and Biochemistry & Molecular Biology, Cumming School of Medicine, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary; 
2 Departments of Clinical Neurosciences and Medical Genetics, Cumming School of Medicine, University of Calgary, Hotchkiss Brain Institute, Alberta Child Health Research Institute.
3 Department of Biochemistry & Molecular Biology, Cumming School of Medicine, Alberta Children’s Hospital Research Institute, University of Calgary.
4 Department of Clinical Neurosciences, Cumming School of Medicine, Hotchkiss Brain Institute, University of Calgary.
* Co-corresponding authors

Institutional affiliation: https://ror.org/03yjb2x39

Author emails: govinda.sharma@ucalgary.ca; rasha.sabouny@ucalgary.ca; matthew.joel@ucalgary.ca; martensk@ucalgary.ca; davide.matino@ucalgary.ca; jdekoning@ucalgary.ca; gerald.pfeffer@ucalgary.ca; timothy.shutt@uclagary.ca

Abstract: Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Here, we describe a novel homozygous MFN2 variant, D414V, in a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy. Characterization of patient fibroblasts reveals phenotypes consistent with impaired MFN2 functions and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

Research Field: Medical and Health sciences

Keywords: mitochondria, MFN2, endoplasmic reticulum, lipid droplets, ataxia, deafness, blindness

Methods: cell culture, microscopy, PCR, oxygen consumption, proximity ligation assay

Funding Information: RS was supported by a Queen Elizabeth II Doctoral Scholarship, an Alberta Graduate Excellence Scholarship, and a University of Calgary Faculty of Graduate Studies Doctoral Scholarship. MJ was supported by a Katharine Sarah Melinda Mei-Ling Thomas Rare Diseases/Biomedical Engineering Research Scholarship and an Alberta Graduate Excellence Scholarship. TS was supported by Canadian Institutes of Health Research.


Usage notes: 

1. Date of data collection (approximate range): 2019-01-01 to 2020-12-31 

2. Geographic location of data collection: Calgary, Alberta, Canada

3. Information about funding sources that supported the collection of the data: 

RS was supported by a Queen Elizabeth II Doctoral Scholarship, an Alberta Graduate Excellence Scholarship, and a University of Calgary Faculty of Graduate Studies Doctoral Scholarship. MJ was supported by a Katharine Sarah Melinda Mei-Ling Thomas Rare Diseases/Biomedical Engineering Research Scholarship and an Alberta Graduate Excellence Scholarship. TS was supported by Canadian Institutes of Health Research.


DATA & FILE OVERVIEW

1. File List: 
Data are contained in five main folders: "Fig 1 (sequencing)", "Fig 2.", "Fig 3.", "Fig 4.", "Fig 5."
* New data for revised manuscript include uncropped western blots for new figure 1E, and revised qPCR data for Fig 3D mtDNA copy number quantification.


Folder name: FIG 1 (sequencing);
	This folder contains 5 files: 
		1. “Control (Forward).ab1”
		2. “Control (Reverse).ab1”
		3. “Mfn2-D414V (Forward).ab1”
		4. “Mfn2-D414V (Reverse).ab1”
		5. “Primers used for sequencing.docx”
	Description and relation between files: The files 1 to 4 are the data files with chromatograms received from the sequencing. The template used for sequencing was whole genome extracted from Control fibroblast (files 1 and 2) and from patient fibroblast with Mfn2-D414V variant (files 3 and 4). File no. 5 contains the sequence of primers used for sequencing. 


Folder name: Fig 1E (western blot)
	This folde contains 1 file
		1. "Fig 1E.pptx"
	Description and relation between files:
		This file, added for the revised manuscript, contains the uncropped western blot images used to generate Fig 1E, including the representative images and quantification of protein expression levels. Cropped regions of interest used to generate the representative figure are indicated.


Folder name: “Fig 2.”

This folder contains two sub folders and a file.
		1. Subfolder name: “2.A. Control”
Description: contains 24 confocal images files (z-stacks in TIF format) of the control fibroblasts, immunostained with anti-TOMM20 antibody. Among these, file named "Cont_23 (rep).TIF" is used as representative image in Figure 2A of the manuscript.

		2. Subfolder name: “2.A. D414V”
Description: contains 26 confocal images files (z-stacks in TIF format) of the Mfn2-D414V variant patient fibroblast, immunostained with anti-TOMM20 antibody. Among these, file named "D414V_1 (rep)_1.TIF" is used as representative image in Figure 2A of the manuscript.

		3. Filename: “2. B. Mito morph quantif”
Description: it is a Graphpad prism file and it contains the data derived from quantification of mitochondrial lengths derived from each of the images in "control" and "D414V" folders; the mitochondrial lengths was determined as follows: Maximum intensity projection of z-stacks were used for analysis. ‘Skeletonize’ function of ImageJ was used to convert image of mitochondria to skeleton. Then ‘Analyze Skeleton’ function of ImageJ was used to find the lengths of the mitochondria. 		
		

Folder name: “Fig 3”
This folder contains two subfolders and a file.
		1. Subfolder name: “Fig 3. A, B and C”
		Contains one subfolder and two files:
			i. Subfolder name: “Fig 3 A.”
				Contains two subfolders
				a. Subfolder name: “Control”
Description: contains 36 confocal images files (z-stacks in TIF format) of the control fibroblast, immunostained with anti-dsDNA (double strand DNA) antibody. Among these, file named "Cont Nucleoid_9(REP).tif" is used as representative image in Figure 3A of the manuscript.

				b. Subfolder name: “D414V”
Description: contains 37 confocal images files (z-stacks in TIF format) of the Mfn2-D414V variant patient fibroblast, immunostained with anti-dsDNA (double strand DNA) antibody. Among these, file named "Cont Nucleoid_9(REP).tif" is used as representative image in Figure 3A of the manuscript.
			ii. Filename: “Fig 3.B. Nucleoid number per cell”
			iii. Filename: “Fig 3.C. Nucleoid size”
Description and relation between files: Above two files (ii and iii) are graphpad prism files that contains the data generated by quantification of images in the subfolder “Fig 3 A.”.  The quantification was performed on the maximum intensity projection of the z-stacks, using ‘Analyze particle’ function of ImageJ software to retrieve the number (contained in file no. ii) and size (contained in file no. iii) of the mitochondrial nucleoids in the image. 

		2. “Fig 3.D. qPCR.xls”
Description and relationship between files: This file contains the raw data from the quantitative real time PCR run with the total DNA extracted from the fibroblasts as a template, 18S rRNA was used as endogenous control, and mitochondrial DNA copy number was evaluated.

		2'. “Fig 3.D. qPCR revised.xls”
Description and relationship between files: This file, with new data for the revised manuscript, contains the raw data from the quantitative real time PCR run with the total DNA extracted from the fibroblasts as a template, 18S rRNA and β-2-Microglobulin were used as endogenous reference to calculate mitochondrial DNA copy number.


		3. “Fig 3. E.”
				i. “Fig 3. E. pptx”
				ii. “Fig 3. E. original file.tif”
Description and relation between files: Above two files (i. and ii) contain the image of Agarose gel electrophoresis of PCR product. File no. ii is the original image file, and the file no. i contains same image where lanes are labelled. Total DNA extracted from fibroblasts was used as template to amplify the mitochondrial DNA using the primers indicated in manuscript. The gel image shown is the PCR product run on 6% Agarose gel. 


Folder name “Fig 4.”
This folder contains two subfolders and two files.
1.	Subfolder name: “Fig 4 A.”
Description: contains 38 confocal images files (z-stacks in TIF format) of the control fibroblast. The signals are from the mitochondria ER contact sites (MERCs) generated by Proximity Ligation Assay (PLA), performed using anti-TOMM20 and anti-Calnexin primary antibodies. Among these, file named "Cont PLA_9(REP).TIF" is used as representative image in Figure 4A of the manuscript.

2.	Subfolder name: “Fig 4 B.”
Description: contains 39 confocal images files (z-stacks in TIF format) of the Mfn2-D414V variant patient fibroblast. The signals are from the mitochondria ER contact sites (MERCs) generated by Proximity Ligation Assay (PLA), performed using anti-TOMM20 and anti-Calnexin primary antibodies. Among these, file named "D414V PLA_33(REP).tif" is used as representative image in Figure 4A of the manuscript.

3.	Filename : “Fig 4 C.”
4.	Filename: “Fig 4 D.”
Description and relationship between files: Above two (file no. 3 and 4) are graphpad prism files that contains the data generated by quantification of images in the subfolders “Fig 4 A.” and “Fig 4 B.”.  The quantification was performed on the maximum intensity projection of the z-stacks, using ‘Analyze particle’ function of ImageJ software to retrieve the number (contained in file no. 3) and size (contained in file no. 4) of the MERCs in the image. 


Folder name “Fig 5.”
This folder contains two subfolders and three files.
1.	Subfolder name: “Fig 5 A. Cont”
Description: contains 36 confocal images files (z-stacks in TIF format) of the control fibroblast, stained with neutral lipid dye (HCS LipidTox Green). Among these, file named "Cont Lipid_23.tif" is used as representative image in Figure 5A of the manuscript.

2.	Subfolder name: “Fig 5 B. D414V”
Description: contains 45 confocal images files (z-stacks in TIF format) of the Mfn2-D414V patient fibroblast, stained with neutral lipid dye (HCS LipidTox Green). Among these, file named "D414V Lipid_2 (REP).tif" is used as representative image in Figure 5B of the manuscript.

3.	Filename: “Fig 5 C. Lipid droplet count per cell”
4.	Filename: “Fig 5 D. Lipid content per cell”
5.	Filename: “Fig 5 E and F. Lipid droplet distances from nuclei”
Description and relationship between files: Above three files (3, 4 and 5) are graphpad prism files that contains the data generated by quantification of images in the subfolders “Fig 5 A. Cont” and “Fig 5 B. D414V.”.  The files also contain the statistical analysis. The quantification was performed on the maximum intensity projection of the z-stacks. 
File no. 3 contains the data derived as follows: individual cell was cropped from the image and using ‘Analyze particle’ function of ImageJ software the number of lipid droplets per cell was retrieved. 
File no. 4 contains the data derived as follows: individual cell was cropped from the image and using ‘Measure’ function of ImageJ the total signal intensity given by the lipid staining was retrieved and indicated as Lipid content per cell. 
File no. 5 contains data derived as follows: individual cell was cropped from the image and using ‘Analyze Particle’ function of the ImageJ software, the co-ordinates of center of lipid droplets and nucleus was determined. Then the distance between the center of lipid droplet to the nucleus was calculated. Fig 5 E is the violin plot that includes the distances of individual lipid droplets from their respective nuclei, in all of the images analyzed. Fig 5 F is the bar graph showing the average distance of lipid droplets from their corresponding nuclei. The individual data points shown are average distance between lipid droplets and the nuclei in one fibroblast.



METHODOLOGICAL INFORMATION

1. Description of methods used for collection/generation of data: 
<Described in the manuscript>

2. Methods for processing the data: 
<Raw data have been submitted; processing for quantification purposes have been discussed wherever applicable>

3. Instrument- or software-specific information needed to interpret the data: 
<Mentioned in the manuscript>

4. People involved with sample collection, processing, analysis and/or submission: Authors


